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| Status |
Public on Jan 10, 2011 |
| Title |
Ini1_ChIPSeq |
| Sample type |
SRA |
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| Source name |
cervix; adenocarcinoma; epithelial
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa S3 cells (ATCC/National Cell Culture Center)
antibody: anti-Ini1 (C-20), Santa Cruz Biotechnology, sc-16189
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| Growth protocol |
HeLa S3 cells were grown in Minimum Essential Medium supplemented with glutamine, 10% FBS, and antibiotics (penicillin-streptomycin) at 37C in 5% CO2, to a density of 6 x10^5 cells/ml.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
HeLa S3 cells were fixed with 1% formaldehyde at room temperature for 10 min and the fixation was terminated by the addition of glycine to a final concentration of 125 mM. The fixed cells were washed in cold 1x Dulbecco’s PBS (Invitrogen) and swelled on ice in 10 ml hypotonic lysis buffer (20 mM Hepes, pH 7.9, 10 mM KCl, 1 mM EDTA, pH 8.0, 10% glycerol, 1 mM DTT, 0.5 mM PMSF, and protease inhibitors). Cell lysates were homogenized with 30 strokes in a Dounce homogenizer. Nuclear pellets were collected and lysed in 10 ml of 1x RIPA buffer per 3 x10^8 cells (1x RIPA buffer: 10 mM Tris-Cl, pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid, 0.5 mM PMSF, 1 mM DTT, and protease inhibitors). Chromatin was sheared with a Branson 250 Sonifier (power setting 2, 100% duty cycle for 7x 30 s intervals) to an average size of less than 500 bp as verified on a 2% agarose gel. Lysates were then clarified by centrifugation for 15 min at 20,000 xg at 4°C. Chromatin immunoprecipitations from 1x10^8 cells were conducted as previously described (Euskirchen et al. 2007). The IgG ChIP DNA samples were run though Qiagen MinElute PCR columns, eluted with 15 μl of Qiagen buffer EB and size-selected (100-350 bp) on 2% agarose E-gels (Invitrogen). Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3' to 5' exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
Raw data from the Illumina Genome Analyzer and Illumina Genome Analyzer II were analyzed with Illumina’s Firecrest, Bustard and GERALD modules for image analysis, basecalling and run metrics, respectively. Replicates were combined for each sample. All reads were aligned against human genome build hg18. ChIP samples were scored against normal IgG (GEO Sample GSM352186) using PeakSeq (see "PeakSeq: Systematic Scoring of ChIP-Seq Experiments Relative to Controls by Rozowsky et al. 2009 Nature Biotechnology). Signal files were created using a 200 bp sliding window for all samples. The PeakSeq output was further filtered using tag count, p-value, excess and enrichment to yield a more stringent set of regions for each factor.
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| Submission date |
Sep 28, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Ghia Euskirchen |
| E-mail(s) |
ghia.euskirchen@stanford.edu
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| Organization name |
Stanford University
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| Department |
Genetics
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| Lab |
Snyder
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| Street address |
1501 S. California Ave.
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| City |
Palo Alto |
| State/province |
CA |
| ZIP/Postal code |
94304 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (2) |
| GSE24397 |
Genome-wide profile of the SWI/SNF chromatin remodeling complex |
| GSE24398 |
Diverse roles and interactions of the SWI/SNF chromatin-remodeling complex revealed using global approaches |
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| Relations |
| SRA |
SRX027270 |
| BioSample |
SAMN00113642 |
| Named Annotation |
GSM601398_Ini1HeLa-peaks-stringent.bed.gz |
| Named Annotation |
GSM601398_Ini1HeLa-peaks.bed.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM601398_Ini1HeLa-peaks-stringent.bed.gz |
226.8 Kb |
(ftp)(http) |
BED |
| GSM601398_Ini1HeLa-peaks.bed.gz |
404.3 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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